| Background: The successful pregnancy involves multiple processes,including embryo implantation,stromal cells decidualization,placenta formation.The abnormality in those processes will result in pregnancy failure.This process requires interactions among several cell types from uterus and fetus,including epithelial cells,stromal cells and maternal decidual cells,as well as inner cells mass and fetal trophoblasts.The maternal-fetal interface serves as an important role in the reproduction system.Lots of studies have been explored the underling mechanism of the maternal-fetal interface formation.The decidualization is an important physiological process of the maternal-fetal interface.The stromal cells arrounding the implantation site undergo extensive proliferation and differentiation after the implantation of embryos.This process provides essential nutrients and energy for embryo development before the formation of placenta.During mice early pregnancy,the primary decidual zone(PDZ)of decidualization is formed on day 5 of pregnancy(D5)and the secondary decidual zone(SDZ)is formed on D6.The decidualization was completed on the day 8 of pregnancy,followed by the development of placenta.The placenta serves as the bridge between the maternal and the fetus,which in vitial for the successful pregnancy.The invasion ability of placental trophoblast was obtained by continuous proliferation and differentiation,which is the foundation of the vascular network of the maternal-fetal interface.The decidualization and the formation of placenta are essential for the successful pregnancy,however,the specific mechanism is not clear.The expression of acyl-Co A binding protein(ACBP)is highly conserved in mammalian.A large number of studies have shown that ACBP was involved in regulating cellular proliferation and apoptosis,fatty acid metabolism,and mitochondrial functions.However,there was no studies to inveatigate the regulation of ACBP during the maternal-fetal interface.Therefore,this present study aims to explore the following questions: 1.Does ACBP participate in mediating decidualization of maternal stromal cells? 2.What is the potential molecule mechanism of ACBP involvement during m ESCs decidualization? 3.Dose SLP2 play a role in regulating the functions of placental trophoblasts?Methods:1.The establishment of several mice models and collection of clinical samples:(1)Mice early pregnancy model: 8-week-old Kunming female mice(25-30g)mated with sexually mature Kunming male mice(25-30g).The day of observation vaginal plug served as the day 1 of pregnancy(D1).Mice were sacrificed by cervical dislocation method to collect the endometrium material on D1,D4,D5,D6 and D8(n=6/group).(2)Mice pseudopregnancy model: 8 weeks old Kunming female mice(25-30g)mated with vasectomized Kunming males' mice to induced pseudopregnancy.The day of observation vaginal plug served as the day 1 of pseudopregnancy(PD1).Mice were killed by cervical dislocation method to collect the endometrium material on PD1,PD4,PD5,PD6 and PD8(n=6/group).(3)Mice embryo delayed and activation implantation model: 8-week-old Kunming female mice(25-30g)mated with Kunming male mice.The day of observation vaginal plug served as the day 1 of pregnancy(D1).The ovaries of pregnancy mice were removed on D3.These mice were given subcutaneous injection of 1mg progesterone(P4)on the morning of pregnancy D4-D6.On day 7,the mice were randomly divided into two groups: one group was subcutaneously injected with 1mg P4.The other group was subcutaneously injected with 1mg of P4 and 25 ng of estradiol(E2).Mice were sacrificed by cervical dislocation method to collect the endometrium material on D8(n=6/group).(4)Mice artificially in vivo induced decidualization model: 8 weeks old Kunming female mice(25-30g)mated with vasectomized Kunming males' mice to induced pseudopregnancy.One side of uterine horn was injected with 10?l of corn oil on PD4(ID),and the other side served as the control(IDC).Mice were killed by cervical dislocation method to collect the endometrium material on PD8(n=10/group).(5)Collection of human samples: The proliferative and secretory phases endometrial samples were collected from women with normal menstrual cycles for surgery or biopsy.All samples were fertile and no hormones treatment within six months.The first-trimester decidua was obtained from women undergoing legally and selectively termination of pregnancy(6-10 weeks).The placental villous tissue(6-10 weeks)was collected from normal pregnant women and patients with unexplained abortion.All samples were obtained with the informed consent of the participants and according to the requirements of the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.2.The expression of ACBP in the endometrium during mice early pregnancy:(1)Mice early pregnancy model: Immunohistochemistry(IHC),Western blot(WB),in situ hybridization(ISH)and RT-q PCR detection of ACBP expression in the endometrium of D1,D4,D5IS(Implantation Site),D5IIS(Inter-Implantation Site),D6 IS,D6IIS and D8.(2)Mice pseudopregnancy model: IHC,WB and RT-q PCR analysis of ACBP expression in the endometrium of PD1,PD4,PD5,PD6 and PD8.(3)Mice embryo delayed and activation model: IHC and ISH were used to detect the expression of ACBP in the uteri of the delayed and induced groups.(4)Mice artificially induced in vivo decidualization model: The uterine weight,morphology,and dtprp m RNA level were used to verify the establishment of mice model.IHC,ISH,WB,and RT-q PCR were applied to detect ACBP expression in the endometrium from control group(IDC)and induced group(ID).(5)Human endometrial specimens: IHC was used to detect ACBP expression in the endometrium of proliferative and secretory phases and deciduama from the first-trimester.3.The molecular mechanism of ACBP regulating decidualization:(1)Mice induced in vitro decidualization: The establishment of induced in vitro decidualization model with the use of the final concentration of 10 n M estradiol(E2)and 1?M progesterone(P4).The model was confirmed by the expression of decidualization markers and the morphology.(2)The regulation of ACBP on the proliferation,apoptosis and senescence of stromal cells: WB,Ed U and Flow Cytometry were used to detect the effect of acbp silencing or CPT1 inhibition on proliferation during the decidualization of mice primary endometrium stromal cells(m ESCs).Flow Cytometry was carried out to detect the effect of acbp silencing or CPT1 inhibition on the apoptosis during m ESCs decidualization.The ?-Galactosidase staining,WB and RT-q PCR were performed to measure the effect of acbp silencing or CPT1 inhibition on the senescence in m ESCs during decidualization.(3)The effect of ACBP on fatty acid and glucose metabolism: WB and RT-q PCR were used to detect the effect of acbp silencing or CPT1 inhibition on the genes expressions involved in fatty acid and glucose metabolism in m ESCs during decidualization.(4)The regulation of ACBP on mitochondrial functions and autophagy: DCFH-DA probe was used to label intracellular reactive oxygen species(ROS)in m ESCs during decidualization.JC-1 probe was employed to determine the mitochondrial membrane potential(MMP)in m ESCs during decidualization.ATP kit was applied to detect intracellular ATP content in m ESCs during decidualization.WB and RT-q PCR were used to detect the expression of genes involved in mitochondrial biogenesis.Mito Tracker Red staining was carried out to detect the morphology of mitochondria in m ESCs during decidualization.WB was applied the measure the expression of autophagy proteins in m ESCs during decidualization.4.ACBP regulated the expression and functions of SLP2 at maternalfetal interface:(1)The regulation of ACBP on SLP2 expression in the maternal-fetal interface: WB and RT-q PCR were applied to measure the effects of acbp silencing on the expression of SLP2 in the endometrial stromal cells(m ESCs)and placental villous cells line(HTR8).(2)IHC was applied to detect the co-localization of ACBP and SLP2 expression in human decidual and villus tissue during the early pregnancy.(3)The regulation of SLP2 on trophoblast proliferation,migration and invasion in HTR8: WB was applied to analysis the effect of slp2 silencing on cells proliferation.Wound heal assay was used to detect the effect of slp2 silencing on the migration.Transwell,WB and villous explant experiments were carried out to assess the effect of slp2 silencing on invasion ability.(4)The regulation of SLP2 on the mitochondrial functions: DCFH-DA probe was used to detect the effect of slp2 silencing on intracellular reactive oxygen species(ROS)production in m ESCs during decidualization.JC-1 probe was applied to determine the effect of slp2 silencing on mitochondrial membrane potential.ATP kit was performed to examine the effect of slp2 silencing on intracellular ATP content.WB and RT-q PCR were applied to detect the effect of slp2 silencing on the expression of mitochondrial biogenesis related genes.(5)IF,WB and RT-q PCR were performed to detect the expression of SLP2 in normal and unknown abortion villous tissue.Results:1.ACBP expression in mice endometrium during early pregnancy:(1)The expression profile of ACBP in the uterus during mice early pregnancy model: IHC results showed that ACBP expressed in D1 endometrial epithelium and glandular cells,but weakly expressed in stromal cells.On D4,ACBP began to expressed in stromal cells.After embryo implantation,the expression of ACBP was detected in the primary decidual zone(PDZ)of D5 and the secondary decidual zone(SDZ)of D6 and D8.ISH results were consistent with the IHC results.The results of WB showed that ACBP protein expressed in D4,D5,D6 and D8 was significantly higher than the expression on D1.On D5 and D6,the expression of ACBP was significantly higher in the implantation site than these in the inter-implantation site.In addition,ACBP protein is highly expressed in D8 endometrium.RT-q PCR results agreed well with WB results.(2)The expression profile of ACBP in mice early pseudopregnancy model: IHC results showed that ACBP expressed in epithelial cells and weakly expressed in stromal cells in PD1.The expression of ACBP was reduced in PD6 and PD8.WB results demonstrated that ACBP was higher expressed in PD4 and PD5 and lower expressed in PD6 and PD8.The results of RT-q PCR were consistent with WB results.(3)The expression of ACBP in mice embryo delayed activation model: IHC and ISH results show that ACBP was mainly expressed in luminal epithelial and glandular cells and weakly expressed in stromal cells in the delayed implantation uterus.After embryo activation,ACBP was strongly expressed in stromal cells.(4)ACBP expression in artificially induced in vivo decidualization model: Morphology of uteri,the uterine weight and the m RNA level dtprp were used to confirm the establishment of model.IHC,ISH,WB and RT-q PCR results showed that ACBP was highly expressed in the induction group(ID)than the IDC.(5)The expression profile of ACBP in human endometrium and the first-trimester deciduama: IHC results indicated that ACBP was weakly expressed in the endometrium of proliferative and secretory phase and strongly expressed in the deciduama of first-trimester.2.The mechanism of ACBP mediating m ESCs decidualization:(1)ACBP regulated CPT1 in the m ESCs: WB and CCK8 results showed that CPT1 was regulated by ACBP in m ESCs.Etomoxir,the inhibitor of CPT1,had no regulation on the expression of ACBP.(2)The effect of acbp silencing or CPT1 inhibition on the decidualization: RT-q PCR and WB results showed that silencing of acbp or CPT1 inhibition reduced dtprp,prl3c1 and hoxa10 expression and affected the morphology during m ESCs decidualization.(3)The effect of silencing of acbp or inhibiting of CPT1 on the proliferation,apoptosis and senescence: Flow Cytometry,WB and Ed U results showed that silencing of acbp gene or inhibiting of CPT1 inhibited cells proliferation.Flow Cytometry and CCK8 results demonstrated that silencing of acbp gene or inhibiting of CPT1 promoted apoptosis.WB,?-galactosidase staining and RT-q PCR results showed that silencing of acbp gene or inhibiting of CPT1 promoted the senescence.(4)The effect of silencing of acbp or CPT1 inhibition on the fatty acid and glucose metabolism: Silencing of acbp or inhibition of CPT1 inhibited the expression of genes involve in fatty acid transport and oxidation,and promoted the expression of genes involve in fatty acid synthesis during decidualization.However,there was no effect of acbp silencing or CPT1 inhibition on the expression of glucose genes during decidualization.(5)The effect of acbp silencing or CPT1 inhibition on the mitochondrial functions during decidualization: The generation of ROS was promoted,as well as mitochondrial membrane potential,intracellular ATP content,mitochondrial biogenesis was inhibited in m ESCs with acbp silencing or CPT1 inhibition during decidualization.The autophagy was induced when m ESCs with the treatment of acbp silencing or CPT1 inhibition.However,acbp silencing or CPT1 inhibition has no effect on the mitochondrial morphology during decidualization.3.ACBP regulated SLP2 expression and functions at maternal-fetal interface:(1)ACBP regulated SLP2 expression: WB and RT-q PCR results show that ACBP regulated SLP2 expression in the maternal-fetal interface.(2)IHC detection of co-localization of ACBP and SLP2 in the serial sections of decidual tissue and placental villous.(3)SLP2 was expressed in CTB,STB and EVT of early pregnancy villi,and is strongly expressed in EVT cell line—HTR8 and HPT8 cells.(4)SLP2 regulated HTR-8/SVneo cells proliferation,migration and invasion: WB,RT-q PCR and CCK8 results showed that silencing of slp2 inhibited the proliferation.The results of wound heal,Transwell,WB,RT-q PCR and villous explant experiments showed that silencing of slp2 inhibited cells migration and invasion.(5)SLP2 regulated mitochondrial functions: The results of DCFH-DA probe,JC-1 probe,ATP content detection kit,WB and RT-q PCR showed that slp2 silencing promoted ROS production,reduced mitochondrial membrane potential,and inhibited mitochondrial biogenesis.However,there was no effect on the morphology of mitochondria.(6)SLP2 expression in aborted villous tissues: IF,WB,and RT-q PCR results showed that both SLP2 m RNA and protein level were down-expressed in aborted villous tissue compared with the normal group.Conclusions:1.The expression of ACBP in mouse early pregnancy model,pseudopregnancy model,embryo delayed activation model and artificial induction in vivo decidualization suggested that ACBP may participate in the decidualization of stromal cells.2.ACBP was involved regulating m ESCs proliferation,apoptosis,senescence,mitochondrial functions and fatty acid metabolism during decidualization.Silencing of acbp or inhibiting of CPT1 impeded m ESCs decidualization.3.ACBP regulated SLP2 expression in the maternal-fetal interface.ACBP and SLP2 were co-localized expressed in several cell types of the maternal-fetal interface.SLP2 regulated proliferation,migration and invasion of trophoblasts through modulating mitochondrial functions. |
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